Lichen compounds that inhibit mycotoxin production

ABSTRACT

Described herein are methods for obtaining useful compounds from lichens.

This application claims benefit of priority to the filing date of U.S.Provisional Application Ser. No. 62/702,424, filed Jul. 24, 2018, thecontents of which are specifically incorporated herein by reference intheir entity.

BACKGROUND OF THE INVENTION

Fungi pose a greater threat to plant and animal biodiversity than othertypes of pathogens (protists, viruses, bacteria, helminths), and thethreat of fungi to ourselves, our food security, and our environment isincreasing (Fischer et al., Nature 484:186-194 (2012)). Human diseasescaused by fungi (mycoses) are on the rise concomitantly with theincrease in numbers of people with immunosuppressive conditions (i.e.AIDS, organ transplants, cancer treatments). Infections ofimmunocompromised humans by fungi including Aspergillus fumigatus andCryptococcus neoformans have increased due to their ability, which isfairly unique among fungi, to grow at body temperatures. Acquired drugresistance in medically relevant microorganisms, including fungi andbacteria, is another severe threat to human health. In addition, fungiand fungal-like organisms are responsible for the destruction of about125 million tons of major crops such as rice, wheat, maize, potatoes,and soybeans each year.

History also shows that fungi and fungal-like organisms can dramaticallyaffect human health and food security. For example, the Irish PotatoFamine, which was caused by the late blight mold, resulted in massstarvation, and the death of 1 million people in the 1840s. Morerecently, a new strain of wheat rust from Africa. UG99 (Uganda 99),which causes up to 100% crop loss, is spreading across wheat growingregions of the world. Mycotoxins pose a threat to human health on nearlyevery continent, and climate change is shifting their appearance to newareas (Magan et al., Plant Pathology 60:150-163 (2011)).

Among mycotoxin contamination of crops, one of the most serious isaflatoxin contamination of crops. Aflatoxin is thought to be the mostpotent naturally occurring carcinogen known. An aflatoxin contaminateddiet has been directly linked with elevated rates of liver cancer,decreased immunity, kwashiorkor, and growth stunting.

In western countries, amounts of aflatoxin contaminants in crops areregulated to below 10 parts per billion. However, outbreaks ofaflatoxicosis are common in underdeveloped countries, mostly amongpoorly nourished rural populations whose staple food is maize. Largelosses of crops contaminated with aflatoxin are common. Worldwide,Aspergillus species cause significant losses in major crops. The annualeconomic impact of aflatoxin contamination on corn and peanutagriculture in the United States is thought to exceed $1 billiondollars.

A second major class of mycotoxins is the trichothecenes, including T-2toxin, nivalenol, deoxynivalenol, and diacetoxyscirpenol, produced bythe fungi Fusarium, Cephalosporium, Myrothecium, Stachybotrys (the blackmold), Trichoderma, and others. In western countries, levels ofdeoxynivalenol are advised to be less than 1 ppm for finished grainproducts for human consumption. The trichothecenes are also damaging tothe health of pigs, cattle, poultry, and particularly swine. Due toclimate change and global warming in recent years, mycotoxigenic fungiare altering their ranges, moving into areas in which they were notpreviously found.

SUMMARY

Described herein are methods that include extracting lichens and testingthe extracts so obtained for their effects on fungal sporulation, fungalhyphal growth and fungal secondary metabolite production. Also describedare anti-fungal compositions, as well as methods for determining themicrobial source and the structure of the active compounds.

One method involves extracting a lichen sample with alcohol, ethylacetate, acetone or a combination thereof to provide a lichen extract,and measuring whether the lichen extract inhibits the growth,sporulation, or mycotoxin production of a fungus.

Another method involves (a) extracting a lichen sample with alcohol,ethyl acetate, acetone or a combination thereof to provide a lichenextract; (b) mixing the extract, a component of the extract, or acompound obtained from the extract with Aspergillus parasiticus strainB62 cells to form an assay mixture; and (c) measuring whether growth,sporulation, or aflatoxin production by the Aspergillus parasiticusstrain B62 cells is different from a control.

Another method involves extracting a lichen as in (a) described above,and (b) mixing the extract, a component of the extract, or a compoundobtained from the extract with Fusarium graminearum to form an assaymixture; and (c) measuring whether growth or deoxynivalenol or 15-acetyldeoxynivalenol or nivalenol production of variations thereof by theFusarium graminearum cells is different from a control.

The methods can be used to provide useful lichen extracts, compounds, orcomponents of the extracts.

Compositions are also described herein that can include a carrier and anextract, compound, or a component of an extract obtained, for example,by the methods described herein.

Such compositions, extracts, compounds, and extract components areuseful for inhibiting fungal growth, fungal sporulation, toxinproduction, or a combination thereof. For example, the compositions,extracts, compounds, and extract components can reduce mycotoxin ortoxin production by at least 10%, at least 20%, at least 30%, at least40%, at least 50%, at least 60%, at least 70%, at least 80%, at least90%, at least 95%, or at least 99%.

The extracts are stable, and retain the ability to inhibit fungalgrowth, fungal sporulation, fungal toxin production, or a combinationthereof for at least ten years, or at least 20 years, or at least 30years.

The compositions, extracts, compounds, and extract components can beapplied to structures, surfaces, agricultural crops, storage bins,storage facilities, animal feed, plant seeds, nuts, plant parts, plantproducts, or a combination thereof. The compositions, extracts,compounds, and extract components can be applied to surfaces inbathrooms, kitchens, closets, basements, attics, entryways, cabinets,boats, barns, animal shelters, warehouses, food storage facilities, andother such surfaces. The compositions, extracts, compounds, and extractcomponents can be applied to grain-producing plants, nut-producingplants, vegetable-producing plants, fruit-producing plants,starch-producing plants, fiber-producing plants, fodder-producingplants, grains, nuts, vegetables, fruits, starch, fibers, flour, fodder,leaves, stock, seeds, oil, or a combination thereof. The compositions,extracts, compounds, and extract components can be applied to almonds,barley, betel nuts, brazil nuts, cashews, chestnuts, coconut, coffee,corn, flour, hazelnuts, macadamia nuts, oats, pecans, peanuts, pinenuts, pistachios, rice, rye, sesame seeds, soybean, spices, walnuts,wheat, or combinations thereof.

Such methods can reduce the mycotoxin, fungal spore, or fungal contentin the surfaces, agricultural crops, storage bins, storage facilities,animal feeds, plant seeds, nuts, plant parts, plant products, or otherplaces.

DESCRIPTION OF THE FIGURES

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1A-ID illustrate some types of lichens and a method for assayinglichens to isolate bioactive lichen compounds with novel activities.FIG. 1A shows an image of Cladonia spp. lichens that grow on the soil.FIG. 1B shows an image of Physica spp. lichens that attach to treebranches. FIG. 1C shows an image of Usnea spp. lichens that attach tobranches. FIG. 1D is a schematic diagram illustrating methods forisolating bioactive lichen compounds with novel activities.

FIG. 2A-2E illustrate high through-put assays in 24 well plates forantifungal effects of lichen extracts on the fungus Aspergillusparasiticus strain B62. The orange-red pigment is a precursor andindicator of aflatoxin production. FIG. 2A shows a 3-day old culture ofAspergillus parasiticus strain B62 that was not treated (control). FIG.2B shows a 3-day old culture of Aspergillus parasiticus strain B62treated with an aflatoxin biosynthesis inhibitor. FIG. 2C shows a 3-dayold culture of Aspergillus parasiticus strain B62 treated with a growthinhibitor. FIG. 2D shows a 7-day old culture of Aspergillus parasiticusstrain B62 illustrating spore production (control). FIG. 2E shows a7-day old culture of Aspergillus parasiticus strain B62 treated with asporulation inhibitor. Plates with 24 wells were used for these assays.

FIG. 3A-3C illustrate that extracts of Cladonia cristatella inhibitsecondary metabolite production by Aspergillus parasiticus strain B62.FIG. 3A shows a culture of Cladonia cristatella mycobiont generated fromascospores. FIG. 3B shows a top view of Aspergillus parasiticus strainB62 cultures illustrating that extracts of Cladonia cristatellamycobiont grown in liquid or solid culture inhibit secondary metaboliteproduction by Aspergillus parasiticus strain B62. FIG. 3B shows a bottomview of Aspergillus parasiticus strain B62 cultures illustrating thatextracts of Cladonia cristatella mycobiont grown in liquid or solidculture inhibit secondary metabolite production by Aspergillusparasiticus strain B62. Arrows indicate the orientation of the assayplate such that the top and bottom of the same well is indicated.

FIG. 4A-4B illustrates that extracts of Evernia prunastri stronglyinhibit hyphal growth and secondary metabolite production by Aspergillusparasiticus strain B62. FIG. 4A shows an image of Evernia prunastri.FIG. 4B illustrates that ethanol extracts of Evernia prunastri inhibitgrowth and aflatoxin accumulation in Aspergillus B62 at 3 differentconcentrations.

FIG. 5A-5B illustrates of the stability of secondary metabolites oflichen species Porpidia (P) and Lecidea (L) as shown by thin layerchromatography (TLC). FIG. 5A shows TLC separation of Porpidia (P) andLecidea (L) extracts collected from an herbarium specimen in 2006. FIG.5B shows TLC separation of Porpidia (P) and Lecidea (L) extractscollected from the same herbarium specimen in 2014. As shown, thesecondary compounds in stored lichens were stable.

FIG. 6 illustrates that extracts of two lichens (Evernia pruastris [1],and Hypogymnia physodes [57]) whose extracts reduced15-acetyl-deoxynivalenol (15-ADON) accumulation in the assay describedfor FIG. 2 in comparison with cultures that were treated with equivalentamounts of solvent (ethanol or acetone).

FIG. 7A-7B illustrate that lichen extracts inhibit deoxynivalenol (DON)accumulation by Fusarium graminearum. FIG. 7A shows that acetoneextracts of lichens Evernia pruastris [1] and Hypogymnia physodes [57]reduced DON production by cultured Fusarium graminearum. FIG. 7B showsthat acetone extracts of lichens Cladonia sylvatica [15], Usnea strigosa[33], Cladonia digitate [58], Ramalina fastigiate [62], Ramalinafraxinea [98], Platismatia glauca [100], Evernia prunastri [112], andCladonia crispata [118] reduced DON production by cultured Fusariumgraminearum.

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

DETAILED DESCRIPTION

As described herein, lichens can produce a plethora of unique secondarycompounds, which are useful in medical, agricultural and food safetyapplications. Lichens exhibit significant longevity and robustnessdespite a close association with diverse microbes and harshenvironmental conditions. Lichens provide model organisms foridentifying and evaluating compounds that can be useful as anti-fungalagents. As described herein, extracts from lichen species were prepared,their activities were tested, and useful compounds have been identifiedand isolated.

Lichens

Lichens are some of the longest-living organisms known. Despite theirslow growth, they very rarely appear die of disease. The lichenizedfungus establishes the main lichen thallus in association with an algaor cyanobacterium, and has been recently shown, a basidiomycete yeast iswidely included (Spribille et al. Science 3S3: 488-492 (2006)). Thisscaffold becomes a niche for a variety of other filamentous ascomycetesand basidiomycetes, yeasts, bacteria, and occasionally insects.

Lichens are defined by symbiotic interactions between a fungus andphotosynthetic partners, green alga or cyanobacterium. Most species offungi, including lichenized fungi, produce a plethora of secondarymetabolites, which are species-specific nonessential metabolites thatare highly diverse and have biological activity. These compounds provideunique functions that can aid organisms in niche-specific adaptationsand can serve as defense against disease and predation.

Antibiotics such as penicillin, and cephalosporin are derived fromfungi, and fungal derived antifungal griseofulvin is used to treatathlete's foot and toe-nail fungus. Other unique fungal metabolites usedin medical applications include: cyclosporins (immunosuppressants),statins (cholesterol lowering drugs) and, fumagillin and pseurotin(angiogenesis inhibitors). One major group of agricultural fungicides,the strobilurins, was originally discovered in extracts of the fungusStrobilurus tenacellus.

The methods described herein can provide new types of compounds withuseful activities. The inventors can use a unique MSU resource, theLichen Herbarium, to identify novel compounds to develop intoanti-fungal products for medicine and agriculture. The lichen collectionin the Plant Biology Department at MSU, an NSF funded herbarium, housesmore than 120,000 specimens and has the greatest taxonomic andgeographical diversity among its collection compared to all other lichenherbaria in North America.

A variety of types of lichens can be used in the methods describedherein and can be a source of useful compounds including, for example,crustose lichens, foliose lichens, leprose lichens, squamulose lichens,and fructose lichens.

Lichens have an algal species associated with a fungal species. Algalspecies associated with lichens are in the genera Nostoc, Trebouxia andTrentephlia, which comprise the algae in 90% of lichens. These algae canbe found free living.

The variation in lichen morphology and physiology is dictated by thefungus. It is therefore most likely that compounds that are uniquelyproduced by a species of lichen are produced by the fungus.

In some cases, the lichen can include species of the genus Acarospora,Arctoparmelia, Amandinea, Aspicilia, Austroparmnelina, Baeonmyces,Biatorina, Buellia, Bryoria, Byssoloma, Calicium, Cladonia, Caloplaca,Candelariella, Cetraria, Cetrelia, Chrysothrix, Cladia, Cladonia,Coenogonium, Collerna, Conotrema, Cryptothecia, Dendrographa,Dermatocarpon, Dictyonema, Diploicia, Diploschistes, Dirinaria,Endocarpon, Flavopanctelia, Flavoparmelia, Flavopunctelia, Fulgensia,Fuscopannaria, Graphina, Graphis, Graphis, Gyrophora, Haematomma,Heppia, Herpothallon, Herpothalon, Heterodea, Heterodermia,Hypocenomyce, Hypogynmia, Lasallia, Lecanora, Lecidea, Leioderma,Lepraria, Letharia, Leptogiun, Lichen, Lecidea, Lichenomphalia, Lobaria,Menegazzia, Metus, Multiclavula, Myelorrhiza, Neophyllis, Nephronma,Niebla, Nostoc, Ochrolechia, Opegrapha, Opegrapha, Pannaria,Paraparmelia, Paraporpidia, Parmelia, Parmeliella, Parmeliopsis,Parmotrema, Peltigera, Peltigera, Peltula, Pertusaria, Pertusaria,Pertusaria, Pertusaria, Pertusaria, Phaeographina, Phaeophyscia,Phlyctis, Physcia, Physconia, Placidium, Placodium, Placopsis,Placopsis, Platismatia, Pseudephebe, Pseudevernia, Pseudocyphellaria,Pseudocyphellaria, Psora, Punctelia, Pyvxine, Ramalina, Rantboldia,Relicina, Rhizocarpon, Rhizoplaca, Roccella, Sagedia, Siphula,Sporopodium, Stereocaulon, Sticta, Stictis, Strigula, Teloschistes,Tephromela, Tlutmnolia, Thysanothecium, Trapelia, Umbilicaria, Usnea,Xanthomaculina, Xanthoparmelia, Xanthoria, or a combination thereof.

Examples of lichens that can be used include those of the type orspecies Acarospora citrina, Arctoparmelia centrifuga, Amandineapunctate, Aspicilia fruticulosa, Aspicilia fruticulosa, Aspiciliavagans, Aspicilia, Austroparmnnelina pruinata, Austroparmelinapseudorelicina, Baeomvces heteromorphus, Biatorina erysiboides, Bryoriafremontii, Buellia frigida, Buellia foecunda, Buellia grimmniae, Buelliasubcoronata, Byssoloma, Calicium chrysocephalum, Caloplaca cinnabarina,Caloplaca cinnabarina, Caloplaca, Candelariella aurella, Cetrariaislandica, Cetrelia olivetorum, Chrysothrix xanthine, Chrysothrix,Cladia aggregate, Cladia moniliformis, Cladia retipora, Cladoniachlorophaceae, Cladonia crispate, Cladonia crispata, Cladoniacristatela, Cladonia didyma, Cladonia digitate, Cladonia fimbriate,Cladonia fimbriate, Cladonia mitis, Cladonia pyxidate, Cladoniapyxidate, Cladonia rangiferina, Cladonia stellaris, Cladonia sylvatica,Cladonia ustulata, Coenogonium implexum, Collema coccophorum, Conotremaurceolatum, Cryptothecia scripta, Dendrographa leucophaea,Dernuatocarpon miniatum, Dictyonenm sericeum, Diploicia canescens,Diploschistes thunbergianus, Dirinaria picta, Endocarpon pusillum,Ephebe lanata, Evernia mesomorpha, Evernia prunastri, Flavopancteliaflaventior, Flavoparmelia caperata, Flavoparmelia rutidota,Flavopunctelia soredica, Fulgensia cranfieldii, Fuscopannarialeucosticte, Fuscopannaria leucosticte, Fuscopannaria subimmixta,Graphis bulacana, Graphina rubens, Graphis mnucronate, Graphis treubii,Gyrophora murina, Haematomma africanum, Haematomma eremaeum, Heppiadespreauxii, Herpothalon rubrocinctum, Heterodea beaugleholei, Heterodeamuelleri, Heterodermia leucomelos, Hypocenomyce australis, Hypogynmiaphysodes, Hypogymnia pulverate, Lasallia papulose, Lasallia pustulata,Lecanora caesiorubella, Lecanora circumborensis, Lecanora conizaeoides,Lecanora epibryon, Lecanora pseudistera, Letharia vulpina, Lethariavulpine, Lecidea lerrena, Leioderma pycnophorum, Lepraria lobfilcans,Lepraria lohiticans, Leptogium saturninum, Lichen caninus, Lichenceuthocarpus, Lichen murorutm, Lichen rufescens, Lichen sylvaticus,Lichenomphalia chromacea, Lecidea psora, Lobaria pulmonaria, Menegazziaplatytrema, Metus conglomeratus, Multiclavula mucida, Myelorrhizaantrea, Neophyllis melacarpa, Nephroma arctica, Nephroma cellulosum,Niebla cephalota, Nostoc, Ochrolechia tartarea, Opegrapha varia,Opegrapha venosa, Parmelia sulcate, Parmeliella plumbea, Pannariasphinctrina, Paraparmelia lithophiloides, Paraporpidia leptocarpa,Parmelia signifera, Parmeliopsis chlorolecanorica, Parmotrenma perlatum,Peltigera canina, Peltigera trumlata, Peltula euploca, Parmelia sulcate,Pertusaria erebescens, Pertusaria pseudodactylina, Pertusariasubventosa, Pertusaria melaleucoides, Pertusaria novaezelandiae,Pertusaria, Phaeographina lamii, Phaeophyscia orbicularis, Phlyctis,Physcia aipolia, Physcia millegrana, Physconia deters, Physciastellaris, Placidiumn squamulosum, Placodium murorum, Placopsisperrugosa, Placopsis, Platismatia glauca, Pseudephebe pubescens,Pseudevernia farinacea, Pseudevernia furfuracea, Pseudocyphellariaberberina, Pseudocyphellaria billardierei, Pseudocyphellaria crocata,Pseudocyphellaria freycinetii, Pseudocyphellaria gilva,Pseudocyphellaria norvegica, Pseudocyphellaria multifida,Pseudocyphellaria gilva, Pseudocyphellaria, Psora crystallifera, Psoradecipiens, Punctelia borreri, Punctelia subrudecta, Pyxine cocoes,Ramalinafarinacea, Ramalina fastigiate, Ramalina fastigiate,Ramalinafraxinea, Ramalina menziesii, Ramalina siliquosa, Ranualinasiliquosa, Caloplaca sp., Tephromela atra, Ramboldia petraeoides,Relicina gemmulosa, Rhizocarpon geographicum, Rhizocarpon geographicum,Rhizoplaca melanophthalna, Roccella canariensis, Roccella portentosa,Sagedia macrospora, Siphula coriacea, Sporopodium vezdeanum,Stereocaulon ramulosum, Stereocaulon saxatile, Sticta limbate, Stictis,Strigula smaragdula, Strigula subtilissima, Teloschistes, Tephromelaatra, Thamnolia vermicularis, Thamnolia vermicularis, Thysanotheciumscutellatum, Thysanothecium sorediatum, Trapelia crystallifera,Trapelia, Umbilicaria decussata, Umbilicaria hyperborean, Umbilicariapustulata, Umbilicaria hyperborean, Usnea antarctica, Usnea arizonica,Usnea himantodes, Usnea inermis, Usnea rubicunda, Usnea scabrida, Usneastrigosa, Usnea strigose, Usnea subfloridana, Usnea wasntathii,Xanthomnaculina convoluta, Xanthoparmelia amplexula, Xanthoparmeliaarapilensis, Xanthoparmelia baeomycesica, Xanthopannelia chlorochroa,Xanthopannelia convolute, Xanthoparmelia cravenii, Xanthoparnmeliaewersii, Xanthoparmelia mougeotina, Xanthoparmelia notlaa,Xanthoparmnelia praegnans, Xanthoparmelia pseudoamphirantha,Xanthopannelia pulla, Xanthoparmelia reptans, Xanthoparmeliasemiviridis, Xanthoparmnnelia substrigosa, Xanthoparmnelia taractica,Xanthopannelia versicolor, Xanthoria filsonii, Xanthoria luhsseana,Xanthoria parietina, or a combination thereof. For example, any of thelichens listed in Appendix I of U.S. Provisional Application Ser. No.62/702,424, filed Jul. 24, 2018 can be used in the methods describedherein and can be a source of useful compounds.

In some cases, lichens such as Cladonia crispata, Cladonia cristatella,Cladonia digitate, Cetraria islandica, Cetrelia olivetorum, Cladoniasylwvaica, Evernia prunastri, Hypogymtnia physodes, Platismatia glauca,Pseudevernia furfuracea, Rantalina fastigiate, Ramnalina fraxinea, Usneastrigosa, or a combination thereof can be extracted and/or can be asource of useful compounds.

Mycotoxin

As indicated above, the most serious mycotoxin may be aflatoxin in termsof contamination of crops. Aflatoxin is mainly produced by strains ofAspergillus flavus and Aspergillus parasiticus, which are ubiquitous innature. Aspergillus flavus and Aspergillus parasiticus have nospecificity towards their hosts and therefore can infect many differentseeds of cereals, nut beans, coffee beans and oil-rich seeds duringcultivation, harvest and post-harvest storage. These fungi infect cropssuch as maize and peanut and produce aflatoxin under the tropical andsubtropical environments. Large amounts of aflatoxins can be produced,especially under humid storage conditions.

Deoxynivalenol is the most common trichothecene affecting agricultureproduction in the US, usually produced by Fusarium graminearum, andcausing major losses in maize, wheat, barley and other small grains. Thepresence of rain during grain flowering encourages fungus dispersal andinfection. Areas of the Midwest and the Red River Valley areparticularly affected by the disease. The presence of the fungus greatlyreduces grain yields, and a very small amount of fungal contaminationrenders barley unusable for malting.

Methods

The activities of lichen extracts were evaluated using experimentalfungal organisms to ascertain whether the extracts could inhibit thegrowth or functions of the fungal organisms. In experiments describedherein tested for their effects on sporulation, hyphal growth andmycotoxin production in the filamentous fungus Aspergillus parasiticus.For example, Aspergillus parasiticus is one of the main producers ofaflatoxin.

Aspergillus parasiticus is useful for screening for antifungal activity.A. parasiticus is a plant pathogen, and produces the mycotoxinaflatoxin. It is extremely closely related to A. flavus, a human andplant pathogen, and A. fumigatus, a human pathogen. Vegetative growthallows fungi to colonize substrates, such as grain, and human tissues inthe case of mycotic disease. Vegetative growth is associated with theproduction of toxins such as aflatoxin. Sporulation is important indissemination of fungi in open air, agricultural fields, and in systemicspread through the human body. Small spores are easily carried in thebloodstream. Thus, compounds effective on A. parasiticus can bepotentially used in medical, agricultural and food safety applications.A primary visual screen of growth, sporulation and secondary metabolismhas been performed on lichen extracts using a mutant of A. parasiticuscalled B62 that accumulates norsolorinic acid, an orange-red precursorof aflatoxin (FIG. 2). As illustrated herein, use of this species forassay screens permits quick identification of lichen extract activities.

Also as illustrated herein, Fusarium graminearum can be used in assaysfor screening lichen extracts for inhibiting deoxynivalenol productionby the Fusarium species.

Lichen extracts can be prepared in a variety of ways. In someembodiments, lichen samples are dried (e.g. at room temperature in thedark). Samples can then be ground in a mill. Samples are then extractedwith a solvent (e.g., an alcohol, ethyl acetate, or acetone). Solventsother than alcohols, ethyl acetate, and acetones were not as effective.The solvent employed for extraction can be methanol, ethanol ethylacetate, acetone, or a combination thereof.

In some embodiments, extracts are then filtered and dried. The resultingresidue can be dissolved in a solvent (e.g., alcohol, ethyl acetate,acetone, water or a combination thereof) to yield a final suspension. Insome embodiments, suspensions are generated with differentconcentrations of lichen material. In some embodiments, the extractpreparation methods described in Examples 1-5 below are utilized.

In some embodiments, active ingredients (e.g., with anti-fungal activityor toxin inhibitory activity) are further purified. Purification methodsare well known in the art and include, but are not limited to,extraction, fractionation, and chromatography. The presence of activeingredient is followed at each step of the process (e.g., using theactivity assays described herein) and fractions with active ingredientsare carried to the next step.

In some embodiments, purified active components are identified. Methodsfor identifying both small molecule and large molecule (e.g., protein)components are available and include, but are not limited to, highpressure liquid chromatography (HPLC), mass spectroscopy, nuclearmagnetic resonance, and combinations thereof.

The experiments described herein illustrate that lichen extracts areuseful as inhibitors of fungal growth and fungal functions. For example,several lichen extracts and pure lichen compounds exhibit inhibitoryeffects on aflatoxin production by Aspergillus parasiticus, when grownin culture or on corn kernels, as a model for field conditions.Specifically, pre-treatment of corn kernels with crude extracts ofCladonia cristatela, Pseudevernia firfuracea, Cetraria islandica,Cetrelia olivetorum, or Evernia prunastri inhibited aflatoxin BIaccumulation when applied prior to inoculation with A. parasiticus.Extracts of C. cristatela showed significant inhibition of AFB I by 99%.Lecanoric acid, identified in the extract of Cetrelia olivetorum, andmethyl-orsellinate, which is present in Physcia stellaris and Hypogymniaphysodes, demonstrated inhibitory effects on growth and aflatoxinproduction in A. parasiticus culture.

Extracts of 70 lichen species were generated. Three of the seventyextracts inhibited Aspergillus parasiticus growth. Ten of the seventyextracts inhibited Aspergillus parasiticus sporulation. Fifty-nine ofthe lichen extracts inhibited secondary metabolite production, whereaflatoxin was used as a proxy for secondary metabolite production. Themost common activity observed for the lichen extracts across thephylogeny was therefore in arresting secondary metabolite production.

Extracts of two lichens were tested in cultures with Fusariumgraminearum, using assay methods like those described for Aspergillusparasiticus. Treated cultures showed significant reduction indeoxynivalenol and 1-acetyldeoxynivalenol without significant effect onFusarium graminearum growth.

Compositions

Compositions described herein can include at least one lichen extract,extract component, or compound purified from a lichen extract. Thecompositions can also include additional components such as a carrier,solvent, surfactant, an additional active ingredient, or a combinationthereof.

These compositions are useful as antibiotics and anti-fungalcompositions.

The composition can contain varying amounts of the extract components,or compounds purified from the extracts described herein. For example,the extracts or compounds can be present in liquid compositions atconcentrations of about 0.1 μg/mL to about 1000 μg/mL, or about 1 μg/mLto about 800 μg/mL, or about 3 μg/mL to about 600 μg/mL, or about 5μg/mL to about 500 μg/mL, or about 5 μg/mL to about 300 μg/mL.

In dry compositions, the extracted components or compounds can bepresent in at weight/weight concentrations of about 0.1 μg/g to about1000 μg/g, or about 1 μg/g to about 800 μg/g, or about 3 μg/g to about600 μg/g, or about 5 μg/g to about 500 μg/g, or about 5 μg/g to about300 μg/g.

The compositions can therefore be dry compositions or liquidcompositions.

In some instances, the extracted components or compounds are dissolvedin a solvent to form a liquid composition with a known concentration ofat least one component or compound from an extract described herein. Thesolvent can be an alcohol, ethyl acetate, acetone, water, or acombination thereof. For example, the solvent can be ethanol methanol,ethyl acetate, acetone, water, or a combination thereof.

The compositions can contain a carrier such as an emulsifier, adispersing agent, thickening agent, a surfactant, a clay, a polymer, acolorant, a wetting agent of ionic or non-ionic type, a natural orregenerated mineral substance, a dispersant, a wetting agent, atackifier, a thickener, a binder, or a mixture of such carriers. Forexample, the compositions can contain polyacrylic acid salts,lignosulphonic acid salts, phenolsulphonic or naphthalenesulphonic acidsalts, polycondensates of ethylene oxide with fatty alcohols or withfatty acids or with fatty amines, substituted phenols (in particularalkylphenols or arylphenols), salts of sulphosuccinic acid esters,taurine derivatives (in particular alkyl taurates), phosphoric esters ofpolyoxyethylated alcohols or phenols, fatty acid esters of polyols, andderivatives of the present compounds containing sulfate, sulfonate andphosphate functions. The presence of at least one surfactant can beincluded when the active compound and/or the inert support arewater-insoluble and when the vector agent for the application is water.For example, surfactant content can be about 5% to 40% by weight of thecomposition.

Coloring agents such as inorganic pigments can be present in thecomposition, for example iron oxide, titanium oxide, ferrocyan blue, andorganic pigments such as alizarin, azo and metallophthalocyanine dyes,and trace elements such as iron, manganese, boron, copper, cobalt,molybdenum and zinc salts can be used. The compounds can be present inpaints along with any available coloring material(s) and othercomponents typically employed in paints.

Optionally, other additional components may also be included, e.g.protective colloids, adhesives, thickeners, thixotropic agents,penetration agents, stabilizers, sequestering agents.

The compositions can also include other ingredients. For example,bactericidal compounds can be employed. In addition, the compoundsdescribed herein can be used together in a composition or they can beused concomitantly with one or more of the other agrichemicals such asvarious pesticides, acaricides, nematicides, other types of fungicides,and plant growth regulators.

Various types of additional fungicides can optionally be included in thecompositions described herein. Examples include copper fungicide such asbasic copper chloride and basic copper sulfate, sulfur fungicide such asthiuram, zineb, maneb, mancozeb, ziram, propineb, and polycarbamate,polyhaloalkylthio fungicide such as captan, folpet, dichlorfluanid,organochlorine fungicide such as chlorothalonil, fthalide,organophosphorous fungicide such as O,O-bis(1-methylethyl)S-phenylmethylphosphorothioate (IBP), edifenphos (EDDP), tolclophos-methyl,pyrazophos, fosetyl, dicarboxylmide fungicide such as iprodionc,procymidone, vinclozolin, fluoromide, carboxyamide fungicide such asoxycarboxin, mepronil, flutolanil, tecloftalam, trichlamide, pencycuron,acylalanine fungicide such as metalaxyl, oxadixyL furalaxyl,methoxyacrylate fungicides such as kresoxim-methyl (stroby),azoxystrobin, metominostrobin, trifloxystrobin, pyraclostrobin,anilinopyrimidine fungicide such as andupurine, mepanipyrim,pyrimethanil, cyprodiniL antibiotic agents such as polyoxin, blasticidinS, kasugamycin, validamycine, dihydrostreptomycin sulfate, propamocarbhydrochloride, quintozene, hydroxyisoxazole, methasulfocarb, anilazine,isoprothiolane, probenazole, chinomethionat, dithianon, dinocap,diclomezine, ferimzone, fluazinam, pyroquilon, tricyclazole, oxolinicacid, iminoctadine acetate, iminoctadine albesilate, cymoxanil,pyrrolnitrin, diethofencarb, binapacryl, lecithin, sodium bicarbonate,fenaminosulf, dodine, dimethomorph, phenazine oxide, carpropamide,flusulfamide, fludioxonil, famoxadone, or combinations thereof. Hence,other types of fungicides can be mixed together with and used in variousamounts with one or more of the extracts or compounds described herein.

The extracts, extract components, and compounds described herein can beused in a weight ratio relative to the other type of fungicide such asfrom 1:0.001 to 1:1000 as a weight ratio. In some instance, the amountof an extract, extract component, or compound purified from a lichenextract relative to the other type of fungicide can vary from 1:0.01 to1:100 as a weight ratio within a composition.

Pesticides can be included in the compositions, with any of thecompounds described herein. The pesticides can include organophosphorouspesticides, carbamate pesticides such as fenthion, fenitrothion,diazinon, chlorpyrifos, ESP, vamidothion, phenthoate, dimethoate,formothion, malathon, trichlorfon, thiometon, phosmet, dichlorvos,acephate, EPBP, methylparathion, oxydemeton-methyl, ethion, salithion,cyanophos, isoxathion, pyridaphenthion, phosalone, methidathion,sulprofos, chlorfevinphos, tetrachlorvinphos, dimethylvinphos,propaphos, isofenphos, ethylthiometon, profenofos, pyraclofos,monocrotophos, azinphosmethyl, aldicarb, methomyl, thiodicarb,carbofuran, carbosulfan, benfuracarb, furathiocarb, propoxur, BPMC,MTMC, MIPC, carbaryl, pirimicarb, ethiofencarb, and fenoxycarb,pyrethroid pesticides such as permethrin, cypermethrin, deltamethrin,fenvalerate, fenpropathrin, pyrethrin, allethrin, tetramethrin,resmethrin, dimethrin, propathrin, phenothrin, prothrin, fluvalinate,cyfluthrin, cyhalothrin, flucythrinate, ethofenprox, cycloprothrin,tralomethrin, silafluofen, brofenprox, and acrinathrin, and benzoylureaand other types of pesticides such as diflubenzuron, chlorfluazuron,hexaflumuron, triflumuron, tetrabenzuron, flufenoxuron, flucycloxuron,buprofezin, pyriproxyfen, methoprene, benzoepin, diafenthiuron,acetamiprid, imidacloprid, nitenpyram, fipronil, cartap, thiocyclam,bensultap, nicotin sulfate, rotenone, mataldehyde, machine oil, andmicrobial pesticides e.g. BT and insect pathogenic virus.

Acricides can be included in the compositions described herein. Theacricides that can be employed include, for example, chlorbenzilate,phenisobromolate, dicofol, amitraz, BPPS, benzomate, hexythiazox,fenbutatin oxide, polynactin, chinomethionat. CPCBS, tetradifon,avermectin, milbemectin, clofentezin, cyhexatin, pyridaben,fenpyroximate, tebufenpyrad, pylidimifen, fenothiocarb, and dienochlor.

As for the aforementioned nematicides, fenamiphos, fosthiazate and thelike can be specifically exemplified; as for plant-growth regulators,gibberellins (e.g., gibberellin A3, gibberellin A4, and gibberellin A7),auxin, 1-naphthaleneacetic acid, and so on can be specificallyexemplified.

More generally, the active compounds can be combined with any solid orliquid additive, which complies with the usual formulation techniques.In general, the composition according to the invention may contain from0.05 to 99% by weight of active compounds, preferably from 10 to 70% byweight.

The extracts, extract component, extracted compounds or compositions canbe provided in a form that is ready-to-use or in a form that can beprepared for use. The extracts, extract component, extracted compounds,or compositions can be applied by a suitable device, such by use of aspraying or dusting device. The extracts, extract component, extractedcompounds, or compositions can be applied by use of brush or roller.

The extracts, extract component, extracted compounds, or compositionscan be provided in concentrated commercial compositions that should bediluted before application to the crop. For example, the extracts,extract component, extracted compounds, or compositions can be providedin dry (e.g., lyophilized) form, or in concentrated form, and thendissolved or diluted as desired. The compositions can be in formulatedinto an aerosol dispenser, as a capsule suspension, as a cold foggingconcentrate, as a dustable powder, as an emulsifiable concentrate, as anemulsion oil in water, as an emulsion water in oil, as an encapsulatedgranule, as a fine granule, as a flowable concentrate for seed or nuttreatment, as a gas (under pressure), as a gas generating product, asgranules, as a hot fogging concentrate, as macrogranules, asmicrogranules, as an oil dispersible powder, as an oil miscible flowableconcentrate, as an oil miscible liquid, as a paste, as a plant rodlet,as a powder for dry seed or nut treatment, as seeds or nuts coated withthe composition, as a soluble concentrate, as a soluble powder, as asolution for seed (or other) treatment, as a suspension concentrate(flowable concentrate), as an ultra-low volume (ULV) liquid, as anultra-low volume (ULV) suspension, as water dispersible granules ortablets, as a water dispersible powder for slurry treatment, as watersoluble granules or tablets, as a water soluble powder for seed or nuttreatment, as a wettable powder, or as a combination thereof (e.g., twotypes of formulations packaged together).

The following Examples illustrate some of the experimental work involvedin the development of the invention.

Example 1: Materials and Methods

This Example describes materials and methods for extraction of lichens.Lichens were collected fresh, and in some cases herbarium specimens weresurveyed, where the oldest herbarium specimen dated back to 1949.Extracts were done in methanol, ethanol, ethyl acetate, and acetone;other solvents did not appear to extract activities as well. Activitieswere found to be quite stable in herbarium specimens, even amongst theoldest specimens.

A primary visual screen of growth, sporulation and secondary metabolismwas performed on lichen extracts using a mutant of Aspergillusparasiticus strain B62 which accumulates norsolorinic acid (NA), anorange-red precursor of aflatoxin. Norsolorinic acid quantification wasperformed by use of a chromameter. Use of this species for initialscreens has facilitated fast identification of activities.

Extracts of two lichens were tested in cultures with Fusariumgraminearum, using methods like the assay methods used for Aspergillusparasiticus. Aspergillus parasiticus was grown on glucose mineral salts(GMS) medium. Fusarium graminearum was grown on High DON Medium fromLinda Harris (McCormick et al., Appl. Environ. Microbiol. 70:2044-2051(2004)).

Example 2: Aspergillus parasiticus Strain B62 Treated with LichenExtracts

A set of high-throughput screens for discovery of antifungal agents weredeveloped using spices as a potential source of inhibitors of mycotoxinbiosynthesis. Black pepper extracts were used in an initial screen.

Extracts of spices were added to Aspergillus parasiticus B62 cultures inGMS, YES, and PMS media (Buchanan & Lewis (1984)) as described in Anniset al. (2000). Changes in growth, sporulation and mycotoxin productionwere observed. Extracts with promising effects were screened using theThin Layer Chromatography (TLC) bioassay developed by the inventors andcoworkers (Annis et al., 2000; Trail et al. 2006). This techniqueresulted in the identification of a novel activity in black pepper thatturned off genes for aflatoxin production but did not arrest fungalgrowth. Based on HPLC, and MS analysis, putative structures weresynthesized and two active structures have been identified. Onestructure inhibits aflatoxin biosynthesis but does not affect fungalgrowth, the other inhibits fungal growth.

In preliminary studies, extracts from four lichen species were testedfor effects on growth, sporulation and mycotoxin biosynthesis in A.parasiticus (FIG. 2). Extracts from one lichen species inhibited fungalgrowth, and extracts from a second lichen greatly reduced aflatoxinaccumulation, but did not affect growth, and inhibited sporulation.These preliminary trials indicate a high frequency of bioactivecompounds in a small group of randomly selected lichens.

High through-put assays were performed in 24 well plates to evaluate theantifungal effects of lichen extracts on the fungal Aspergillusparasiticus strain B62. An orange-red pigment was a precursor andindicator of aflatoxin production.

FIG. 2A-2E illustrate high through-put assays in 24 well plates forantifungal effects of lichen extracts on the fungus Aspergillusparasiticus strain B62. FIG. 2A shows a 3-day old culture of Aspergillusparasiticus strain B62 that was not treated (control). FIG. 2B shows a3-day old culture of Aspergillus parasiticus strain B62 treated with anaflatoxin biosynthesis inhibitor. FIG. 2C shows a 3-day old culture ofAspergillus parasiticus strain B62 treated with a growth inhibitor. FIG.2D shows a 7-day old culture of Aspergillus parasiticus strain B62illustrating spore production (control). FIG. 2E shows a 7-day oldculture of Aspergillus parasiticus strain B62 treated with a sporulationinhibitor. Plates with 24 wells were used for these assays.

Example 3: Assay Results

Table 1 shows results of assays for inhibition of growth, aflatoxinproduction, and sporulation of Aspergillus parasiticus B62 by extractsof the indicated lichen species are shown. The growth, aflatoxinproduction, and sporulation of Aspergillus parasiticus B62 was comparedto Control. The symbols used in Table 1 were: N—No effect; SI—StrongInhibition (90-100%); I—Inhibition (38-80%); A—Activation (˜150%);SA—Strong Activation (>150%).

TABLE 1 Effects of Lichen Extracts on growth, aflatoxin production, andsporulation of Aspergillus parasiticus B62 Lichen species GrowthAflatoxin Sporulation Bryoria fremontii N SI N Cetraria islandica N SI NLatvia May 2016 (I Fract) Cetrelia olivetorum CA Bay Area August 2016Cetrelia sp CA Bay Area August 2016 Cladonia chlorophaceae N I NPellston MI 3 Oct. 2015 Cladonia crispata Pellstone MI 17 Sep. 2016Cladonia cristatela (British soldier) N SI N Frances 17 Nov. 2015 RedCaps/Rotten Cart Cladonia didyma + N SI A Cladonia fimbriata Cladoniadigitata N SI N Latvia, Lielupe, 9 Aug. 2015 Cladonia mitis Pellstone MI17 Sep. 2016 Cladonia pyxidata (Owen) N I N Cladonia rangiferina N I ALatvia May 2016 Ropazi Maris Small quantity Cladonia stellaris N SI IYoshkar-Ola 2009 Cladonia sylvatica I SI I Yoshkar-Ola 2009 Dendrographaleucophaea N SI N f. minor Frances March CA 2017 Dermatocarpon miniatumN SI N S Dakota Black Hills on rock Herbarium Ephebe lanata N SI N FloraSuecica 1961 Herbarium Evernia mesomorpha N SI N? Pellston (#3) 3 Oct2015 Evernia prunastri SI SI SI Yoshkar-Ola 2009/GB 2015 Flavopancteliaflaventior N SI I by Green House 19 Jun. 2015 Flavoparmelia caperata NSI I by Green House 19 Jun. 2015 Flavopunctelia soredica N SI N Quercusalba tree, F.T., 8 Aug 15 Fuscopannaria leucosticta N N N N Carolina1967 Herbarium Graphis bulacana N SI N West Indies Trinidad 1963Herbarium Herpothalon rubrocinctum 2016 Hypogymnia physodes I SI SILatvia, Lielupe, 9 Aug. 2015 Lecanora caesiorubella CA March 2017Lecanora circumborensis CA March 2017 Lecidea psora N SI Wyoming 1956Herbarium Lepraria lohiticans N “Amorphic” Dust Lichen Pellston MI 3 Oct2015 Leptogium saturninum N I S Dakota Alan 1961 Letharia vulpina, N SIN wolf lichen, Alan, 1994 Lobaria pulmonaria Maine Frances 10-15 Jul.2016 N Nephroma arctica N SI MSC0066318 Niebla cephalota N SI N Asilomar2015 Parmelia sulcata + N SI N Flavoparmelia caperata Parmeliellaplumbea N SI N Canary Islands Tenerife 1964 Herbarium Peltigera canina NI/N N Pellston MI 3 Oct. 15, (#10) Peltigera trumlata AF10981 N I NPeltula euploca N SI A Colorado Rabit Mt 1960 Herbarium Pertusariaerebescens N N N East Falklands Kidney Is1, 1968 Herbarium Phaeophysciaorbicularis, Alan N I N Physcia aipolia Okemos 2014 N SI N Physciamillegrana N SI SA Frances' Yard 22 Jun. 2015 Physcia stellaris N SI Iby Vet Clinic 19 Nov. 2015 Physconia deters N I I MI Alan 8 Oct. 1976Platismatia glauca N SI I Latvia May 2016 Ropazi Maris/Ilze Pseudeverniafarinacea N(I/SI SI N Latvia May 2016 Ropazi Maris/Ilze Fracts) (P.furfuracea by ITS/NCBI) Pseudevernia furfuracea I SI N Latvia, Lielupe,9 Aug 2015 Pseudocyphellaria berberina N N SI AF10717 Pseudocyphellariacrocata AF 10876 N SI N Pseudocyphellaria freycinetii AF N SI I(?) 10716Pseudocyphellaria gilva, AF 10877 N SI N Pseudocyphellaria norvegica AFN I N 10878 Ramalina farinacea Asilomar March N N N 2015 Ramatinafastigiata N SI N Latvia, Lielupe, 9 Aug. 2015 Ramalina fraxinea LatviaMay 2016 N I I Carnicava Skaidrite Ramalina menziesii Asilomar 2015 N SIA(one well) Rhizoplaca melanophthalma N N N S Dakota Black Hills 1960Herbarium Roccella portentosa N SI N Chile Alan 14 Nov. 1965Stereocaulon saxatile MI Cheboygan N SI N County 1949 HerbariumThamnolia vermicularis N SI N British Columbia 1970 HerbariumUmbilicaria hyperborea + N I N Pseudephebe pubescens Usnea arizonicaMaine July 2016 Frances Usnea rubicunda N N N Asilomar March 2015 Usneastrigosa N I N Usnea subfloridana N SI N Usnea wasmathii SI Latvia May2016 Ropazi Maris/Ilze Small quantity Xanthoparmelia chlorochroa A SIXanthoria hasseana N I SA yellow Pellston (#3) 3 Oct. 2015 Xanthoriaparietina N SI N?

Example 4: Cladonia cristatella Extracts Inhibit Secondary MetaboliteProduction

This Example illustrates that extracts of Cladonia cristatella inhibitssecondary metabolite production by Aspergillus parasiticus strain B62.

Ascospores from Cladonia cristatella were isolated and cultured asillustrated in FIG. 3A. Colonies from these colonies were extracted withethanol and acetone. Aspergillus parasiticus strain B62 was culturedwith the extracts. As illustrated in FIG. 3B, the extracts inhibitedsecondary metabolite production by Aspergillus parasiticus strain B62.

Example 5: Evernia prunastri Extracts Inhibit Hyphal Growth, SecondaryMetabolite Production, and Sporulation

This Example illustrates that extracts of Evernia prunastri inhibithyphal growth, secondary metabolite production, and sporulation byAspergillus parasiticus strain B62.

Extracts of Evernia prunastri were cultured with Aspergillus parasiticusB62 in GMS (Buchanan & Lewis, 1984). As illustrated in FIG. 4B theEvernia prunastri extracts exhibited strong inhibitory activity againsthyphal growth, secondary metabolite production and sporulation byAspergillus parasiticus B62. Purification of the active compound andX-ray crystallography determined that evemic acid was present in activefraction of Evernia prunastri extracts, where the evemic acid structureis shown below.

Example 6: Lichen Extracts Inhibit Accumulation of Aflatoxins in CornKernels

The following lichen extracts were tested for their capacity to reduceaflatoxin accumulation in corn kernels.

#1 Evernia prunastri

#81 Cladonia cristatella

#99 Pseudevernia farinacea

#101 Cetraria islandica

#129 Dendrographa leucophaea f. minor

Extracts of lichens #1, 99 and 101 demonstrated protective effectsagainst aflatoxin contamination, showing 80-70% inhibition of aflatoxinaccumulation on DeDell field corn kernels (200 spores/kernel, 96 h postinoculation). Extracts obtained from lichen #81 inhibited aflatoxin B1accumulation by 99% on sweet corn kernels. The extract from #129 wasactive on Monsanto field corn kernels after 72 h post inoculation. Allof these represent statistically significant findings.

Example 7: Stability of Compounds in Lichen Material During Storage andUse of Herbarium Specimens for Extraction

Chemicals preserved in dry lichens appear to be largely stable understandard herbarium storage conditions. Thin layer chromatography (TLC)analyses of acetone extracts from the identical lichen specimensobtained eight years apart demonstrated similar metabolite profiles(FIG. 5A-5B).

Example 8: Lichen Extracts Reduce 1-Acetyldeoxynivalenol by Fusariumgraminearum

Extracts of two lichens. Evernia pruastris and Hypogymnia physodes, weretested in cultures with Fusarium graminearum, using assay methods likethose used for the assays of Aspergillus parasiticus.

As illustrated in FIG. 6, the Fusarium graminearum cultures treated withthese lichen extracts (Evernia pruastris [number 1], and Hypogymniaphysodes [number 57]) showed significant reduction in1-acetyldeoxynivalenol production without significant effects on growth.

Example 9: Extracts of Various Lichen Reduce Deoxynivalenol Production

The fungus Fusarium graminearum is a plant pathogen which causesfusarium head blight, a devastating disease on wheat and barley. ThisExample illustrates that extracts from various lichens can reducedeoxynivalenol (DON) production by Fusarium graminearum.

In a first experiment. Fusarium graminearum cultures were grown in24-well plates for 6 days in the dark at room temperature. Each wellcontained 1 ml High DON Medium (McCormick et al., 2004) plus 20 μl oflichen extract (either Evernia pruastris [number 1] or Hypogymniaphysodes [number 57]), while control wells contained 20 μl ethanolwithout a lichen extract. Cultures exhibiting decreased production ofred/pink pigment but no effects on growth were analyzed for DONaccumulation. FIG. 7A shows the effects of these lichen ethanol extractson DON accumulation by Fusarium graminearum grown in culture for 6 days.

In a second experiment. Fusarium graminearum cultures were grown in24-well plates for 6 days in the dark at room temperature. Each wellcontained 1 ml High DON Medium plus 20 μl of a lichen extract fromCladonia sylvlaica [15], Usnea strigosa [33], Cladonia digitate [58],Ramalina fastigiate [62], Ramalina fraxinea [98], Platismatia glauca[100], Evernia prunastri [112], or Cladonia crispata [118]. Controlwells contained 20 μl acetone without any lichen extract. Culturesexhibiting decreased production of red/pink pigment but no effects ongrowth were analyzed for DON accumulation.

As shown in FIG. 7B acetone extracts of lichen extracts from Cladoniasylvlaica [15], Usnea strigosa [33], Cladonia digitate [58], Ramalinafastigiate [62], Ramalina framinea [98], Platismatia glauca [100],Evernia prunastri [112], and Cladonia crispala [118] all reduced DONproduction by Fusarium graminearum.

Example 10: Extracts of Various Lichen Remain Stable for Decades

This Example described results of assaying various lichens that had beenstored for extended periods of time to determine if they could stillinhibit growth, norsolorinic acid (NA) production, and sporulation byAspergillus parasiticus.

Lichens were obtained after storage under herbarium storage conditions,extracts were made, and the extracts were added to Aspergillusparasiticus cultures.

This assessment of lichen extract activity was performed in 2014-2015.

Table 2 shows which lichens were evaluated, and whether the Aspergillusparasiticus grew, accumulated norsolorinic acid, and sporulated (Nindicates that no growth, no NA, or no sporulation was observed).

TABLE 2 Activity of lichen compounds to inhibit Norsolorinic Acidaccumulation is stable for decades under Herbarium storage conditions.Inhibition of Lichen/ Norsolorinic ID Year collected/ acid numberStorage facility Growth accumulation Sporulation 85 Stereolaulonsaxatile, N 90-100% N Michigan Cheboygan County 1949, MSU Herbarium 87Petula euploca, N 90-100% N Colorado Rabbit Mt, 1960, MSU Herbarium 88Graphis bulacana, N 90-100% N West Indies, Trinidad, 1963, MSU Herbarium90 Ephebe lanata, Flora N 90-100% N Suecica, 1961, MSU Herbarium 93Lecidea psora, N 90-100% N Wyoming, 1956, MSU HerbariumActivity of assay was performed in 2014-2015. N, no effect.

As illustrated, each of the lichens shown in Table 2 inhibited 90-100%of norsolorinic acid accumulation even though the lichens had beenstored since 1949-1963.

REFERENCES

-   Annis S L. Velasquez L, Xu H, Hammerschmidt R. Linz J, Trail F,    2000. J Agric Food Chenm. 48(10):4656-60.-   Buchanan R L and Lewis D F. Appl. Environ Microbiol. 48(2):306-10    (1984).-   Butler M S, Fontaine F, and Cooper M A, 2013. Planta Med. Epub ahead    of print. Georg Thieme Verlag KG Stuttgart, N.Y.-   Cheon. DM. Jang D S, Kim H Y, Choi K S, Choi S K, 2013. Korean J.    Microbiol. Biotechnol. 49(2): 165-171.-   Fisher M C. Henk D A, Briggs C J. Brownstein J S, Madoff L C, McCraw    S L. Gurr S J, 2012. Nature 484:186-194.-   Gaffoor I, Brown D W, Plattner R. Proctor R H. Qi W, Trail F, 2005.    Euk Cell. 4:1926-1933-   Gao L, Cai M Shen W. Xiao S, Zhou X and Y Zhang, 2013. Microbial    Cell Factories 2013, 12:77.-   Kim E S. Kap S C, and Sang K C. 2012. Korean J. Microbiol.    Biotechnol. 40 (1): 23-29.-   Kupferschmidt K. 2012. Science 337:636-638.-   Magan N, Medina A, Aldred D. 2011. Plant Pathology 60:150-163.-   Miao V. Coeffet-LeGal M-F. Brown D, Sinnemann S. Donaldson G,    Davies J. 2001. TRENDS in Biotechnology 19(9): 349-355.-   Mitrović T, Stamenković S. Cvetković V, Tošić S, Stanković M,    Radojević I. Stefanović O, Čomić L, Dačić D, {umlaut over    (C)}určić M. and Marković S. 2011. Int. J. Mol. Sci. 12, 5428-5448.-   Roze L V, Beaudry R M, Arthur A E, Calvo A M, and JE Linz. 2007. App    Environ Microbiol. 73(22): 7268-7276.-   Roze L V, Koptina A V, Laivenieks M, Beaudry R M. Jones D A,    Kanarsky A V. Linz J E. 2011. Appl Microbiol Biotechnol.    92(2):359-70.-   Rugbjerg P, Naesb M. Uffe H Mortensen U H, and Frandsen R J N.    Microbial Cell Factories 2013, 12:31 Shrestha G. and St. Clair L L.    2013. Phytochem Rev 12:229-244.-   Trail F. Hammerschmidt R, Linz J E. Xu, H, Velasquez L.    Annis S. 2004. U.S. Pat. No. 6,825,216.-   Trail F. Hammerschmidt R. Linz J E, Xu, H. Velasquez L,    Annis S. 2006. U.S. Pat. No. 7,041,678.-   Zambare V P and Christopher L P. 2012. Pharmaceutical Biology 50(6):    778-798.-   Wiemann P. and Keller N P. 2013. J Ind Microbiol Biotechnol. Epub    ahead of print.

All patents and publications referenced or mentioned herein areindicative of the levels of skill of those skilled in the art to whichthe invention pertains, and each such referenced patent or publicationis hereby specifically incorporated by reference to the same extent asif it had been incorporated by reference in its entirety individually orset forth herein in its entirety. Applicants reserve the right tophysically incorporate into this specification any and all materials andinformation from any such cited patents or publications. The followingstatements describe some of the elements or features of the invention.Because this application is a provisional application, these statementsmay become changed upon preparation and filing of a nonprovisionalapplication. Such changes are not intended to affect the scope ofequivalents according to the claims issuing from the nonprovisionalapplication, if such changes occur. According to 35 U.S.C. § 111(b),claims are not required for a provisional application. Consequently, thestatements of the invention cannot be interpreted to be claims pursuantto 35 U.S.C. § 112.

Statements:

-   -   1. A method comprising extracting a lichen sample with alcohol,        ethyl acetate, acetone or a combination thereof to provide a        lichen extract, and measuring whether the lichen extract        inhibits the growth, sporulation, or mycotoxin production of a        fungus.    -   2. The method of statement 1, wherein the fungus produces        aflatoxin.    -   3. The method of statement 1 or 2, wherein the fungus is an        Aspergillus or a Fusarium.    -   4. The method of statement 1, 2, or 3, wherein the fungus is an        Aspergillus parasiticus.    -   5. The method of statement 1-3 or 4, wherein the fungus is        Aspergillus parasiticus strain B62.    -   6. The method of statement 1-4 or 5, wherein the fungus is        Fusarium graminearum.    -   7. The method of statement 1-5 or 6, wherein the alcohol is        methanol or ethanol.    -   8. The method of statement 1-5 or 6, wherein the lichen sample        is extracted with acetone.    -   9. The method of statement 1-7 or 8, further comprising        purification of a compound that inhibits the growth,        sporulation, or mycotoxin production of a fungus from the lichen        extract to provide a purified compound.    -   10. The method of statement 1-8 or 9, further comprising        determining a structure of a compound that inhibits the growth,        sporulation, or mycotoxin production of a fungus from the lichen        extract to provide a compound with a structure.    -   11. The method of statement 1-9 or 10, further comprising        determining an amount of the purified compound or the compound        with the structure that inhibits the growth, sporulation, or        mycotoxin production of a fungus.    -   12. The method of statement 1-10 or 11, further comprising        applying the lichen extract, the purified compound, or the        compound with the structure to a surface, an agricultural crop,        a storage bin, a storage facility, an animal feed, a plant seed,        a nut, a plant part, a plant product, or a combination thereof.    -   13. The method of statement 1-11 or 12, wherein the surface is a        bathroom, a kitchen, a closet, a basement, an attic, an        entryway, a cabinet, a boat, a barn, an animal shelter, a        warehouse, a grain storage compartment, or a food storage        facility surface.    -   14. The method of statement 1-12 or 13, wherein the plants        comprise grain-producing plants, nut-producing plants,        vegetable-producing plants, fruit-producing plants,        starch-producing plants, fiber-producing plants,        fodder-producing plants, grains, nuts, vegetables, fruits,        starch, fibers, flour, fodder, leaves, stock, seeds, oil, or a        combination thereof.    -   15. The method of statement 1-13 or 14, wherein the plant        products are almonds, barley, betel nuts, brazil nuts, cashews,        chestnuts, coconut, coffee, corn, flour, hazelnuts, macadamia        nuts, oats, pecans, peanuts, pine nuts, pistachios, rice, rye,        sesame seeds, soybean, spices, walnuts, wheat, or combinations        thereof.    -   16. The method of statement 1-14 or 15, wherein the mycotoxin        content in a surface, an agricultural crop, a storage bin, a        storage facility, an animal feed, a plant seed, a nut, a plant        part, a plant product, or a combination thereof is reduced by        spraying a composition onto the plants or plant products.    -   17. The method of statement 1-14 or 15, wherein the mycotoxin        content is reduced by at least 10%, at least 20%, at least 30%,        at least 40%, at least 50%, at least 60%, at least 70%, at least        80%, at least 90%, at least 95%, or at least 99%.    -   18. The method of statement 1-16 or 17, wherein the lichen is        any of the lichens described in this application.    -   19. The method of statement 1-17 or 18, wherein the lichen is        Cladonia crispata. Cladonia cristatella, Cladonia digitate,        Cetraria islandica, Cetrelia olivetorum, Cladonia svlvatica,        Evernia pnsnastri, Hypogyrnia physodes, Platismatia glauca,        Pseudevernia fifuracea, Ranalina fastigiate, Ramalina fraxinea,        Usnea strigosa, or a combination thereof.    -   20. An extract obtained by the method of statement 1-18 or 19.    -   21. An extract component or compound obtained by the method of        statement 1-19 or 20.    -   22. An extract comprising lichen compounds solubilized in        alcohol, ethyl acetate, acetone or a combination thereof.    -   23. A composition comprising a carrier and an extract, compound,        or a component of an extract obtained by the method of statement        1-18 or 19.    -   24. The composition of statement 23, where the carrier is a        solvent or surfactant.    -   25. The composition of statement 23 or 24, where the carrier is        a solvent selected from methanol, ethanol ethyl acetate, or        acetone.    -   26. The composition of statement 23-24 or 25, where the carrier        is an emulsifier, a dispersing agent, a thickening agent, a        surfactant, a clay, a polymer, a colorant, a wetting agent, a        mineral substance, a dispersant, a tackifier, a thickener, a        binder, or a mixture of such carriers.    -   27. The composition of statement 23-25 or 26, wherein the        compound(s) are at a concentration of 0.1 μg/mL to about 1000        μg/mL, or about 1 μg/mL to about 800 μg/mL, or about 3 μg/mL to        about 600 μg/mL, or about 5 μg/mL to about 500 μg/mL, or about 5        μg/mL to about 300 μg/mL.    -   28. The composition of statement 23-26 or 27, wherein the        composition comprises weight/weight concentrations of one or        more compounds at about 0.1 μg/g to about 1000 μg/g, or about 1        μg/g to about 800 μg/g, or about 3 μg/g to about 600 μg/g, or        about 5 μg/g to about 500 μg/g, or about 5 μg/g to about 300        μg/g.    -   29. The composition of statement 23-27 or 28, wherein the        compound(s) are at a concentration of about 0.1 ppm to 500 ppm,        or about 1 ppm to 400 ppm, or about 2 ppm to 300 ppm, or about 5        ppm to 250 ppm, or about 10 ppm to 150 ppm, or about 12 ppm to        100 ppm, or about 15 to 50 ppm, or about 20 ppm to 35 ppm, or        about 25 ppm.

What is claimed:
 1. A method comprising (a) extracting a lichen sample with alcohol, ethyl acetate, acetone or a combination thereof to provide a lichen extract; (b) mixing the extract, a component of the extract, or a compound obtained from the extract with Aspergillus parasiticus cells or Fusarium graminearum cells to form an assay mixture; and (c) measuring whether growth, sporulation, or mycotoxin production by the Aspergillus parasiticus cells or Fusarium graminearum cells is different from a control.
 2. The method of claim 1, wherein the control is an average amount of growth, sporulation, or mycotoxin production by Aspergillus parasiticus cells or Fusarium graminearum cells that were not mixed with the extract, the component of the extract, or the compound obtained from the extract.
 3. The method of claim 1, wherein the assay mixture comprises a culture medium that promotes mycotoxin production by the Aspergillus parasiticus cells or the Fusarium graminearum cells.
 4. The method of claim 1, further comprising isolating an extract component or compound that reduces the growth, sporulation, or mycotoxin production by the Aspergillus parasiticus cells.
 5. The method of claim 1, further comprising isolating an extract component or compound that reduces the growth, sporulation, or mycotoxin production by the Fusarium graminearum cells.
 6. The method of claim 1, wherein the cells are Aspergillus parasiticus strain B62 cells.
 7. The method of claim 1, further comprising isolating a component or compound from the lichen extract.
 8. The method of claim 7, further comprising mixing the component of the extract, or a compound obtained from the extract with Aspergillus parasiticus cells or Fusarium graminearum cells to form an assay mixture; and measuring whether growth, sporulation, or mycotoxin production by the Aspergillus parasiticus cells or Fusarium graminearum cells is different from a control.
 9. The method of claim 1, further comprising applying the lichen extract, the component of the extract, or a compound obtained from the extract to structure, to a surface, to an agricultural crop, to a storage bin, to a storage facility, to an animal feed, to a plant seed, to a nut, to a plant part, to a plant product, or to a combination thereof.
 10. The method of claim 1, wherein toxin content of a surface, an agricultural crop, a storage bin, a storage facility, an animal feed, a plant seed, a nut, a plant part, a plant product, or a combination thereof is reduced by applying a composition of the lichen extract, the component of the extract, or the compound obtained from the extract to the surface, agricultural crop, storage bin, storage facility, animal feed, plant seed, nut, plant part, plant product, or the combination thereof.
 11. The method of claim 10, wherein the mycotoxin content is reduced by at least 50%.
 12. An extract obtained by the method of claim 1 that reduces mycotoxin content by at least 50%.
 13. A composition comprising a lichen extract, a component of a lichen extract, or a compound obtained from a lichen extract, wherein the lichen extract is a lichen extracted by an alcohol, ethyl acetate, acetone or a combination thereof. 